Development and Characterisation of SGLT2 Monoclonal Antibodies

Abstract

Background: Chronic kidney disease (CKD) often leads to renal fibrosis, a condition marked by excessive extracellular matrix (ECM) deposition that disrupts kidney function and can result in end-stage renal disease (ESRD). The sodium-glucose cotransporter 2 (SGLT2), highly expressed in renal tubules, contributes to glucose reabsorption and is associated with CKD through its role in metabolic dysregulation and renal fibrosis. Targeting SGLT2 offers a tissue- specific approach for delivering therapeutic agents to reduce fibrosis. Current treatments lack tissue specificity and can cause systemic side effects, highlighting the need for targeted therapeutic strategies. Aims: To generate and characterise an SGLT2-targeting monoclonal antibody for reducing renal fibrosis in CKD and explore its therapeutic mechanism. Methods: Mice were immunised with an SGLT2-diphtheria toxin A (DTA) fusion protein. DTA was used to break immune tolerance against the self-protein SGLT2 by eliciting a stronger immune response. Spleen cells were harvested, RNA extracted, and cDNA created to generate a phage display library of fragment antigen-binding (Fab) antibody regions. Biopanning isolated high-affinity antibodies, followed by competitive ELISA to determine the binding epitopes. Full-length SGLT2 was transiently transfected into mammalian cells for validating antibody specificity through western blotting, while immunohistochemistry (IHC) on mouse kidney sections confirmed tissue-specific binding. Results: Several SGLT2-targeting Fab fragments were identified through phage display. Competitive ELISA and western blotting confirmed antibody specificity with minimal cross- reactivity. IHC demonstrated kidney-specific binding, while biotinylated Fab fragments exhibited high affinity and stability. The monoclonal antibody could be further developed to deliver anti-fibrotic agents to SGLT2-expressing renal tissues, potentially reducing ECM deposition and preserving kidney function. Conclusions: Monoclonal antibody fragments specifically targeting SGLT2 were successfully generated, suggesting a potential therapeutic approach for treating renal fibrosis in CKD. Further, in vivo studies are needed to explore their efficacy.